Following vascular injury, adhesive ligands engage integrin aIIbb3 to effect platelet aggregation and spreading. These responses are triggered by clustering of integrin complexes, which initiates "outside-in" signals" to reorganize the actin cytoskeleton. Recent evidence implicates the Syk tyrosine kinase in early events of outside-in signaling. The goal of this project is to understand the molecular basis of signal relay from aIIbb3 and Syk to actin. Specific Aim 1 will test the hypothesis that outside-in signaling is initiated by a direct interaction between Syk and the cytoplasmic tail of beta-3. This interaction and its molecular basis will be evaluated in cells using bioluminescence resonance energy transfer, and the effects of other beta-3 tail-binding proteins on the interaction will be assessed. Human and gene-targeted murine platelets will be used to determine how hIIbb3 regulates Syk and whether Src family kinases are required. Specific Aim 2 will test the hypothesis that adapter molecular, particularly SLP-76 and SLAP-130, propagate signals from aIIbb3 and Syk to actin. Co-precipitation studies will assess the composition of aIIbb3-based signaling complexes that form when fibrinogen binds to platelets. Then, the functions of SLP-76 and SLAP- 130 in aIIbb3 signaling will be determined with retroviral reconstitution studies in SLP-76 (-/-) and SLAP-130 (-/-) mice, and in a mutational analysis of these adapters in CHO cells. Particular attention will focus on SLP-76 or SLAP-130 binding partners implicated in regulating actin organization, including Nck/PAK and VASP. Since some Syk effectors may reside within lipid rafts, biochemical and confocal analyses will explore whether outside-in signaling in platelets is influenced by perturbations of these membrane microdomains. Taken together, these studies will provide the details of a road map from aIIbb3 to actin that is relevant to hemostasis. Furthermore, they may lead to identification of new drug targets and serve as a paradigm for integrin signaling in both blood cells.